The broad long-term objective of this proposal is to characterize possible mechanisms involved in the initiation of biosynthesis/release of the endocannabinoid anandamide. Anandamide has been shown to display the same physiological effects as plant derived cannabinoids by acting as an agonist at the brain and peripheral cannabinoid receptors (CB1 and CB2 respectively). A better understanding of the mechanisms that initiate the synthesis/release of anandamide may reveal new drug targets that provide therapeutic benefits in multiple conditions. Interestingly, anandamide has been recently shown to exhibit endovanilloid activity by activating the Ca2+-permeable vanilloid receptor (VR1), a member of the TRP family of receptors. The proposed studies examine the cellular localization of VR1 receptors and their potential link to anandamide synthesis/release. The hypothesis to be explored is that VR1 receptors are found in the lipid raft/caveolae domains of the plasma membrane and that their activation by noxious stimuli, and possibly anandamide itself, acts to stimulate the synthesis/release of anandamide from caveolae. The Specific Aims are (1) To determine if the VR1 receptor is localized to a specific domain of the plasma membrane and (2) To determine if VR1 receptor activation will stimulate the synthesis/release of anandamide from caveolae. Several TRP channels have been shown to localize to the caveolin-rich domains of cells, and evidence suggests that the precursors for anandamide are enriched in caveolin-rich membranes. The Research Design will seek to show that VR1 receptors are localized in the caveolae domains of the plasma membrane using subcellular fractionation techniques, Western blot analysis, and immunofluorescence. A novel use of the fluorescent calcium biosensor, yellow cameleon proteins will be exploited as a molecular marker of VR1-induced Ca 2. level increases in caveolae/lipid raft domains in living cells. The ability of VR1 to stimulate the synthesis/release of anandamide in a Ca2*-dependent fashion will be measured by quantification of anandamide accumulated in either the subcellular fractions or the assay buffer following VR1 stimulation. The use of the emerging technology of fluorescent biosensors to explore yet unidentified mechanisms associated with endocannabinoid biosynthesis will provide new opportunities to understand the cellular biology of the system modified by cannabinoids.